CT Technology

Question 1

Transfection is the process of introducing nucleic acids into cells by non-viral methods. Transduction is the process whereby foreign DNA is introduced into another cell via a viral vector.

Question 2

A common way to validate that a genetic material was successfully introduced into cells is to measure protein expression. This is typically performed by Western blot or immunostaining

Question 3

Transfection is the process of deliberately introducing naked or purified nucleic acids into eukaryotic cells

Question 4

Transfection uses chemical and non-chemical based methods to transfer foreign DNA into the cells.

Question 5

Nucleofector technology enables highly efficient, transfection of primary cells, stem cells, neurons, and cell lines that have traditionally been difficult to transfect via electroporation and other non-viral transfection methods.

Question 6

The Nucleofector® Technology uses a specific combination of optimized electrical parameters and cell type-specific solutions which enables transfer of a molecule directly into the cells’ nucleus

Question 7

Three specialized components are key to the successful and efficient transfection of primary cells or cell lines using a variety of substrates:

A Nucleofector® Device that comprises unique electrical parameters pre-programmed for each optimized cell type, to deliver the substrate directly into the cell nucleus and the cytoplasm. The different platforms we offer provide different specifications for various applications.

Question 8

Cont’d:

Nucleofector® Kits, containing dedicated Nucleofector® Solutions and Supplements. These act as a protective environment for high transfection efficiency and cell viability, while maintaining physiologically relevant cellular conditions. Specified Nucleofection vessels, pipettes, and a fluorescent positive control vector (pmaxGFPTM Control Vector) are also provided.

Optimized protocols offering comprehensive guidance for optimal Nucleofection® Conditions along with tips for cell sourcing, passage, growth conditions and media, and post-transfection culture.

Question 9

Nucleofector features/benefits:

• High transfection efficiencies of up to 90% for plasmid DNA and 99% for oligonucleotides, like siRNA.
• Excellent preservation of the physiological status and viability of transfected cells.
• Analysis of transfection results already shortly after transfection possible.
• Easy to use technology, with over 650 cell-type specific protocols that have led to direct transfection success.
• Transfection of a wide range of substrates, including DNA, mRNA, miRNA, siRNA, peptides or proteins.
• Transfection of hard-to-transfect cells, including primary cells, stem cells, neurons and cell lines, as well as cells in adherence.

Question 10

ACT = Adoptive Cell Transfer
TIL = Tumor Infiltrating Lymphocytes
TCR = T-cell receptors
CAR = Chimeric Antigen Receptors

Question 11

Cellectis gene-edits “Chimeric Antigen Receptor” (CAR) T-cells from healthy donors into “off-the-shelf” immunotherapy product candidates that are designed to work for the largest number of patients

Question 12

Gene Editing

Gene insertion
- Insertion is used to add a new function to the genome. For example in drug discovery, or in order to overcome a genetic defect like hemophilia.
Gene correction
-Correction is used to replace an existing defective sequence (which generally impacts the gene’s functions) with a functional sequence. For example, to treat a serious genetic disease such as cystic fibrosis.
Gene inactivation
-Inactivation is used to prevent the expression of a gene. This approach can be used to treat persistent viral infections such as AIDS.

Question 13

Most of the trials conducted to date have used CD19-targeted CAR T cells. But that’s changing quickly, in part out of necessity. Some patients with ALL don’t respond to the CD19-targeted therapy. In those who experience a complete response, up to a third will see their disease return within a year. Many of these disease recurrences have been linked to ALL cells’ no longer expressing CD19, a phenomenon known as antigen loss.

Question 14

A single-chain variable fragment (scFv) is not actually a fragment of an antibody, but instead is a fusion protein of the variable regions of the heavy (VH) and light chains (VL) of immunoglobulins, connected with a short linker peptide of ten to about 25 amino acids

Question 15

The pioneering CAR T therapies initially approved for clinical use are technically limited. They have a single-purpose scFv receptor, no way to control the dose of any given cell, and no mechanism to address tumor heterogeneity or antigen loss. All of these issues can cause CAR T therapies to fail – either during treatment or due to relapse and antigen loss after administration

Question 16

Compared with traditional pharmaceuticals, the clinical development time for these cell therapies is much shorter, leaving very little time for process development, or chemistry, manufacturing, and controls (CMC). That means that manufacturing decisions must be made early, process translation must be quick, and the system must be scalable from Phase 1 trials to commercial manufacturing.

Question 17

The antigen binding domain is the portion of the CAR that confers target antigen specificity.
Several characteristics of the scFv impact CAR function beyond simply recognizing and binding the target epitope. For instance, the mode of interaction among the VH and VL chains as well as the complementarity-determining regions’ relative positions impact the affinity and specificity of the CAR for its target epitope

Question 18

Limitations of CAR-T cell therapy
Antigen escape
One of the most challenging limitations of CAR-T cell therapy is the development of tumor resistance to single antigen targeting CAR constructs. Although initially single antigen targeting CAR-T cells can deliver high response rates, the malignant cells of a significant portion of patients treated with these CAR-T cells display either partial or complete loss of target antigen expression. This phenomenon is known as antigen escape.

Question 19

One of the challenges in targeting solid tumor antigens is that solid tumor antigens are often also expressed on normal tissues at varying levels. Therefore, antigen selection is crucial in CAR design to not only ensure therapeutic efficacy but also to limit “on-target off-tumor” toxicity.

Question 20

The costimulatory domain offers another modifiable region in CAR design that can be tailored based on tumor type, tumor burden, antigen density, target antigen–antigen binding domain pair, and concerns of toxicity. Specifically, 4-1BB domains result in a lower risk of toxicities, higher T cell endurance, and a lower peak level of T cell expansion, while CD28 co-stimulatory domains are associated with CAR-T cell activity that is more rapid in onset and subsequent exhaustion.

Question 21

Apheresis professionals use processed total blood volume (TBV) as a key parameter for apheresis collections. They determine processing volume targets based on the donor’s size, sponsor’s requested cell count and product volume targets.

Question 22

Apheresis centers use validated SOPs for any processing services they offer, such as cryopreservation. Sponsor-required deviations from those validated procedures may require revisions to the center’s SOPs along with validation of the new procedure prior to use. In addition, centers will require training on your protocol-specific steps.

Question 23

The maintenance of cells in culture for any period of time places selective pressures on the cells that are different from those in vivo. Cells in culture age and may accumulate both genetic and epigenetic changes, as well as changes in differentiation behavior and function. Scientific understanding of genomic stability during cell culture and assays of genetic and epigenetic status of cultured cells are still evolving.

Question 24

Antigen escape and lack of CAR T-cell persistence are the most common causes of relapse after CAR T-cell therapy. CAR T-cell products currently approved by the FDA are considered “second generation” because they include an additional costimulatory domain such as 4-1BB or CD28 to improve persistence and potency.1 To further augment the antitumor activity, a third-generation CAR is being developed with multiple costimulatory domains and a fourth-generation CAR contains a transduction domain to promote production of a T-cell−activating cytokine such as interleukin (IL)-1.

Question 25

The CAR molecule consists of 3 parts: (1) an extracellular, antibody-derived, single-chain variable fragment to bind a specific antigen on the tumor cell with a hinge region, (2) a transmembrane domain (part of CD3, CD8, CD28, or FcεRI) and (3) an intracellular domain, consisting of the intracytoplasmic activating domain (CD28, CD27, CD134, CDB7, or CD3ζ) derived from the T-cell receptor with or without a second costimulatory factor (CD28 or 4-1BB) (Figure 14). CAR T cells bind antigens independent of human leukocyte antigens (HLAs), an important feature because HLA downregulation is a key mechanism of cancer cell escape from immune surveillance.

Question 26

Manufacturing CAR T cells takes 2 to 4 weeks, during which time the cells undergo gene modification, expansion ex vivo, and freezing for subsequent re-infusion. The patient-specific CAR T-cell product is then transferred back to the hospital and is re-infused into the patient.6 Patients receive lymphodepleting chemotherapy to enhance CAR T-cell expansion, proliferation, and persistence. Patients typically receive lymphodepleting chemotherapy with fludarabine and cyclophosphamide 2 to 14 days before receiving CAR T-cell infusion.3,8 After CAR T-cell infusion, the chimeric receptor recognizes an antigen leading to effector cell activation, proliferation, and a milieu of cytokine release including IL-6, soluble IL-6 receptor, soluble IL-2Ra, interferon gamma (IFN-g), and granulocyte-macrophage colony-stimulating factor (GM-CSF).

Question 27

Collected T cells must retain the ability to respond to stimulation signals, successfully undergo transduction (vector entry, reverse transcription, and integration), and ultimately function when reinfused. However, the composition of autologous starting material from patients with cancer is highly variable, influenced by patient age and defects due to underlying disease or pretreatment with lymphotoxic agents. For instance, memory T-cell concentrations in patients with ALL and non-Hodgkin lymphoma decrease with each course of standard-of-care treatment

Question 28

Cryopreservation of the apheresis product after collection can also influence cell quantity and quality

The presence of certain cellular subsets at culture initiation can negatively impact T-cell activation and expansion. The presence of MDSCs and monocytes can hamper the ex vivo activation and expansion of T cells.

Question 29

Another target cell purification method involves enriching for T cells or specific T-cell subsets using antibody-conjugated magnetic beads for positive or negative selection, which may improve product purity and yield and, potentially, clinical responses. A few centers have focused on enriching T cells based on the expression of CD62L, CD4, and CD8, as these subsets may improve persistence or antitumor activity.

Question 30

Whether a selection process is employed or not, a T-cell activation step is required for adequate transduction and expansion. To achieve this goal, T cells are activated via polyclonal stimulation using soluble anti-CD3 antibodies or immobilized CD3 and CD28 antibodies (22, 60–63). Immobilized anti-CD3 antibodies provide better cross-linking and activation of T cells, while CD28 antibodies activate costimulatory pathways in the target cells.

Question 31

Paramagnetic beads, such as Dynabeads, can also be coated with these antibodies; in suspension, the coated beads provide appropriate stimulation for much larger T-cell cultures. Prior to formulating the final cellular products, the beads need to be removed, as they could pose a hazard if infused into the patient. Removal is achieved by disrupting the T-cell/bead aggregates via agitation and then passing the suspension through a strong magnetic field, which retains the beads but allows cells to flow through.

Question 32

Recently, stimulation reagents such as Transact have been employed, which utilizes humanized anti-CD3 and anti-CD28 antibodies conjugated to a colloidal polymeric nanomatrix. The nanomatrix can be washed out in a centrifugation step, prior to final product formulation. Another similar method of T-cell stimulation is using a hydrogel “stimulation matrix” incorporating antibodies, which can also be removed by washing after stimulation and expansion.

Question 33

Besides the challenges of obtaining pure and viable starting material, significant batch-to-batch variability exists with autologous cells. Most current manufacturing protocols rely on open, manual processing steps susceptible to operator-introduced errors and contamination, and not easily amenable to scale-out.

Question 34

Open systems for T-cell cultures are easily adaptable to CAR T cell expansion. Familiarity and ease of use of T flasks, gas-permeable culture bags, and membrane bioreactors have made such systems an appealing choice for small- to medium-scale manufacturing processes. For example, in the G-Rex bioreactor, T cells are cultured on a gas-permeable membrane located on the bottom of the culture “bottle”. This bioreactor can be placed in a regular lab incubator; for media changes and cell harvests, the bioreactor is opened in a biosafety cabinet, to manually pipette fluids and cells in and out under aseptic conditions.

Question 35

Recently, a functionally closed version of this system was developed, allowing for a peristaltic pump to move fluids and cells in and out of the bioreactor. A single unit of this bioreactor can provide a 500 cm2 gas-permeable surface area with 5 L media capacity, which is sufficient to expand large numbers of CAR T cells for clinical applications. The Xuri cell expansion system is another adaptable and functionally closed process for CAR T cell manufacturing based on the WAVE bioreactor platform, which also employs a separate cell washing unit. However, both the G-Rex and the Xuri systems require skilled operators at all stages.

Question 36

The Quantum hollow fiber bioreactor platform has been used as a functionally closed system for cell expansion.. Initially designed for adherent cell culture, this bioreactor has a total surface area of 2.1 m2 per disposable cartridge and has been adapted for the expansion of cells in a suspension culture. Further studies are necessary to investigate the Quantum system for genetically modified CAR T cell expansion.

Question 37

To achieve commercial-scale production of CAR T cell therapies, end-to end automation is desirable. There are currently two approaches to automation: fully automated closed systems or partially automated systems. Fully automated closed processes eliminate any handling of the product during manufacturing but are restricted to a single product in each production run. Fully automated systems such as the CliniMACS Prodigy (Miltenyi Biotec) and the Cocoon (Lonza) are capable of isolating T cells (either CD3+ or CD4/CD8 enriched) from an apheresis product and moving them into a functionally closed culture and transduction process

Question 38

To overcome GvHD issues in allogeneic protocols, gene-editing methods such as CRISPR-Cas9, transcription activator-like effector nucleases (TALEN), or zinc finger nucleases (ZFN) have been used to disrupt the expression of endogenous T-cell receptors and/or MHC on the allogeneic T cells. While these promising approaches mitigate immunogenic recognition of MHC-deficient CAR T cells by host CD8 T cells, the infused cells become more prone to natural killer–cell killing

Question 39

The tolerable number of potentially alloreactive CAR T cells infused into the recipient has yet to be established. Several allogeneic CAR T cell products have been tested in clinical trials, with the most recent inducing remission in patients with relapsed and/or refractory B-ALL. As expected, however, allogeneic CAR T cell persistence remains shorter as compared with autologous approaches.

Question 40

Allogeneic T cells, just as autologous cells, must be expanded without compromising in vivo function. T-cell exhaustion and culture-associated metabolic changes restrict the number of population doublings, overall culture times, and quantities. Overcultured cells lose in vivo efficacy and show hallmarks of the “exhausted” phenotype.

Question 41

Most manufacturers characterize the final CAR T cell product by flow cytometry, with many characterization panels currently in use. Several ranges/measures exist to determine the total T-cell number via CD3 expression; to characterize the CD4+ and CD8+ subsets within this population (54); to detect transduced and not transduced cells; and to identify any contaminating cell subsets, such as monocytes/macrophages.

Answer 41

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Question 42

The early stage of the current clinical trials mean that target cell numbers have not been established. Therefore, the scale of the process is still not defined.

Question 43

T cell therapy starts with obtaining the patient’s WBCs by leukapheresis, an apheresis method that separates white blood cells from whole blood. The blood components are usually separated by density with continuous or intermittent centrifugation methods using density gradient media.

Question 44

Anticoagulants added during the apheresis process, red blood cells and platelets are contaminations which are usually removed in a washing step. Anticoagulants potentially alter the behaviour of the cells during activation

Question 45

Red blood cells can influence clinical efficacy and platelets can lead to clumping of the cells. To remove red blood cells and platelets, manual Ficoll density gradient centrifugation is applied in early reports and again in more recent clinical trials . Alternatively, automated cell-washers such as the COBE 2991 Cell Processor (Terumo BCT, Lakewood, CO, USA), the Haemonetics CellSaver (Braintree, MA, USA), the Biosafe Sepax II or the monocyte depleting CaridianBCT Elutra are used. After washing, the WBCs are either directly used or frozen in controlled rate freezers such as the Cryomed.

Question 46

Several methods that mimic the natural stimulation of T cells (TCRs) have been developed and implemented. A common approach is to add OKT3 (anti-CD3 monoclonal antibody (mAb)) and interleukin (IL) 2. Simultaneous co-culture with irradiated healthy donor peripheral blood mononuclear cells (PBMCs) and lymphoblastoid cell lines (LCL; human Epstein-Barr-virus (EBV) infected PBMCs) is sometimes referred to as the ‘rapid expansion protocol’. The concentration of IL-2 used varies considerably from one trial to the next.

Question 47

A clinical trial used anti-CD3/CD28 antibody coated magnetic beads as artificial antigen presenting particles and found that activation with these allows for engraftment of cells that retain their memory phenotype more than with OKT3/IL-2. The superparamagnetic beads have a diameter of 4.5 μm and are efficiently removed with a strong electromagnet, leaving <100 residual beads per 3 × 106 cells at the end of production.

Question 48

During expansion. the beads were used to continuously stimulate the cells. Cytokine production was 10-100 fold higher suggesting that activation is stronger using bead-activation compared to other methods such as activation with anti-CD3 antibodies and IL-2

Question 49

Antibody-coated paramagnetic beads also present several processing advantages. By magnetically retaining the beads bound with cells, cell culture steps such as washing and enrichment are more easily facilitated. The beads can be used for selection and activation of the cells without the need to remove them until harvest. Perfusion or media exchange is possible without losing great amounts of expensive stimulating antibodies since they are coupled to the beads. It has been shown, that activation with anti-CD3/CD28 beads results in less exhausted and thus more persistent T cells than activation with OKT3 (anti-CD3 mAb) and IL-2.

Question 50

Gene delivery can be divided into viral and non-viral methods. In CAR T cell therapy, electroporation of naked DNA, plasmid-based transposon/transposase systems and viral vectors, in particular retro- or lentiviruses have been applied for gene delivery.

Question 51

High efficiency transduction using retro- or lentiviruses requires activation of the T cells. Especially in retroviruses, that only transduce dividing cells, proliferation is essential for gene delivery. With both approaches, there is a risk of oncogenic gene insertion, while lentiviral integration is theoretically less prone.

Question 52

Viral gene delivery methods require packaging cell lines for the cGMP production of the viral vector. This is labour intensive and expensive mainly because vector production has to be carried out in a separate clean room facility and additional vector release testing has to be performed. Lentiviral vectors are typically produced via transient transfection using large amounts of plasmid DNA; making them more expensive than retroviral vectors that can be produced using stable packaging cell lines.

Question 53

A relatively new method of CAR gene delivery is the use of transposon/transposase systems. A transposon is a sequence of DNA with the ability to change position within a genome via transposase excision and insertion [35]. The CAR transgene can be inserted into a transposon sequence on a plasmid, with the transposase encoded either within the transposon or separately. Plasmids are electroporated into T-cells prior to activation, where then the transposase excises the CAR-containing-transposon and inserts the sequence into the T-cell genome.

Question 54

The variety of methods in the expansion process can be divided into three approaches. First, plates or T-flasks. This approach has a high requirement of trained operators manufacturing the product in an open-handling manner in safety cabinets usually using multiple flasks/ plates per product. Tissue culture flasks and plates are used for smaller patient cohorts. Alternatively, static culture bags, or eventually scaled up to static culture bags after initiating expansion in flasks or plates. Scaling up using larger static culture bags instead of increasing the number of flasks or plates has a number of advantages. Bags can be connected in a sterile way reducing the amount of open-handling steps. The third approach utilizes the most advanced technology. Starting in bags or flasks, the expansion finally takes place in a RM bioreactor that runs in perfusion.

Question 55

The combination of bead activation, viral transduction and expansion in the RM bioreactor is most common production method for activation, gene delivery, and expansion. The second most frequent approach is activation with mAbs/IL-2, viral transduction and expansion in flasks. This approach is the most manual and unautomated production strategy. Third most used is the combination of bead activation, viral transduction and expansion in static culture bags.

Question 56

Off-tumor on-target toxicities are potential fatal risks whe testing novel receptors. Solutions to mitigate these risks include messenger RNA (mRNA) transfection of the T cells, so that they express the CAR only in a transient manner limiting the effect of CAR toxicity issues. nother design includes a drug dependent “kill switch” for inducible apoptosis, useful for mitigating long-term off-tumor toxicities such as B cell aplasia in CD19-CARs.

Question 57

Although methods are available to test potency of the T cells, no standardized method is used, making it difficult to compare CAR T cell potency across studies or platforms. To date, potency assays are one of the main challenges in CAR T cell product characterization during QC. This is mainly due to the complexity of the mode of action of CAR T cell therapies and the lack of standardized methods.

Question 58

Automated cell therapy manufacturing devices can enable manufacturing of a large number of personalised CAR T cell therapies. Researchers have used the CliniMACS Prodigy, an automated cell therapy production platform, for the generation of clinically relevant numbers of CD19-CAR T cells. They showed that the man hours needed for manufacturing can be greatly decreased with this system compared to a production process with the rocking motion bioreactor.